The over arching hypothesis is that the brain is an important reservoir for retroviruses because of their ability to infect long lived terminally differentiated cells and viral products released from these cells can cause immune activation and neuronal injury Aim 1: To understand the mechanism of viral persistence in brain If there is any hope to eradicate HIV, close attention to the viral reservoirs in the brain is necessary. The brain is a unique site of viral latency since it infects resident macrophages/microglia and astrocytes. These cells have very low turnover rate, and the mechanism of viral entry and persistence is very different than that of lymphocytes which are the major cell type infected by the virus in the lymphoid organs. Our laboratory has focused its efforts on studying the mechanism of viral infection of astrocytes. We have found that while free viral particles can enter these cells, cell to cell contact with lymphocytes is the most efficient way to infect astrocytes. We have discovered that the virus enters astrocytes by using CXCR4 and is aided by formation of tight junctions between the cells which we have termed, viral synapses.Another interesting observation made in our laboratory is that astrocytes express CD4 on the cell surface when eposed to cytokines which makes them vulnerable to HIV infection. This helps explain why astrocytes in vivo are infected in large numbers yet these cells have been very difficult to infect in vitro with cell free virus. We have also found that upon entry, the virus can enter the endolysosomal pathway which acts as a host defense mechanism. Hence strategies than modulate these pathways could have a significant effect on the establishment of a reservoir in the brain. We are now confirming these findings using virus and lymphocytes from CSF of HIV infected individuals to determine if there are starins of HIV that preferentially infect astrocytes. Two manuscripts are currently being prepared that detail these findings. However the turnover rate of the cells in the brain (microglia and astrocytes) is also critical to the eradication of the reservoir, hence we have initated a study in a mouse model of inflammation to determine if the turnover rate of these cells may be altered during the state of inflammation. preliminary studies suggest that the turnover rate is accelerated in specific areas within the brain. Aim 2: To investigate the mechanism of neuronal injury by HIV and endogenous retroviruses Despite the use of antiretroviral agents and excellent control of the virus in the periphery, HIV infected patients continue to develop cognitive impairment. Currently available antiretroviral agents have no effect on the production of early viral proteins once the virus has integrated into the chromosome. One of these proteins, Tat, has been shown to be neurotoxic. Our laboratory was one of the first to demonstrate its toxic potential and we are now investigating the mechanisms by which it causes neurotoxicity. We have found that the protein can cause synaptic injury at very low concentrations without causing neuronal death. We have characterized the proteins and the morphological changes at the level of the dendrites in human neurons and are further investigating the underlying mechanisms. Using a similar approach we are investigating the mechanisms by which the envelop protein of an endogenous retrovirus-K causes neurotoxicity. We have cloned the gene into an expression vector, created a transgenic line that expresses the protein and have found that the mice develop a motor neuron disease simialr to ALS. The mice have been extensively characterized by behvioral testing and histopathological studies. We are now determinign the mechanism by which HERV-K is regulated in neurons and if HERV-K can be transmitted from one cell to another. Aim 3: To develop therapeutic approaches to prevent viral activation and formation of viral reservoirs in the brain. We are generating cell lines with inducible expression of HIV-Tat protein and the HERV-K virus. These cell lines will be used in high through put screening assays to screen for anti-sense molecules and for small pharmacological compounds that suppress their production. Currently viral constructs are being prepared, which will be cloned into appropriate vectors for transfection of cell lines. In summary, we have shown that astrocytes in the brain are an important reservoir for HIV and that cell to cell contact with lymphocytes is necessary for viral entry and the lysosomal pathway in these cells regulates the intracellular trafficking of the virus and its ultimate ability to successfully infect these cells. Further, we have shown that the HIV protein Tat and the env protein of endogenous retrovirus-K are neurotoxic and we are now studying the underlying mechanisms involved in these effects. Finally, we have also discovered that the Tat protein of HIV can stimulate T cells in a T cell receptor independent manner using a unique mechanism. We will now develop therapeutic strategies for preventing the activation of these genes.